Microscopes applying pulsed laser beams are well-known in the art, particularly, confocal microscopes using two or more pulsed laser beams of distinct wavelengths. The two or more laser pulses are temporarily and spatially synchronized and are of different wavelengths. The laser pulses have to arrive at a particular scanning spot on a sample to be imaged either simultaneously or with a specific time delay between the laser pulses of different wavelengths, depending on the particular type of microscope. Examples for microscopes applying two different laser pulses are for instance a coherent anti-Stokes Raman scattering (CARS) microscope, a stimulated Raman scattering (SRS) microscope, a Raman-induced Kerr-effect scattering (RIKES) microscope, a sum-frequency generation (SFG) microscope, and a stimulated emission depletion (STED) microscope. An example for a microscope applying three distinct wavelengths that are incident on the sample with clearly pre-defined time delays or overlap is for instance non-degenerate anti-Stokes Raman scattering (CARS) microscope.
For various applications of these types of microscopes, it is desirable to select the transmitted wavelength of one or more of the laser pulses of distinct wavelengths. It is well known that acousto-optic tunable filters (AOTFs) can be used for selecting wavelengths. A radiofrequency is applied to the AOTF crystal transmitting the respective laser pulse of a particular wavelength, and by changing the radiofrequency only a particular wavelength of the laser light that correlates to the particular, changed radiofrequency is transmitted through the crystal.
One specific property of AOTFs is that laser pulses of different central wavelengths propagating through the AOTF experience different time delays, i.e. propagate at different speeds through the AOTF. In other words, two laser pulses of different wavelengths experiencing different effective refractive indices hence emerge at different times from the AOTF. The amount of temporal “walk-off” depends on the wavelength separation between the two laser beams. For the above-mentioned microscopes, two or more pulsed laser beams, however, have to arrive at a given time delay or temporal overlap at the sample to generate a strong signal. This time delay can be either zero or non-zero, but has to be specific for each particular application. Wavelength differences may range from several nanometers to hundreds of nanometers. Tuning at least one of the wavelengths results in a change of the temporal delay between the pulses and therefore may result in non-optimal imaging if the resulting time delay is non-optimal.